Isoflavones are compounds having an isoflavone skeleton which are contained predominantly in leguminous plants. It is known that isoflavones will act as a phytoalexin in plants. A phytoalexin is an antibacterial substance which plants produce against stresses such as microorganism infection.
Moreover, isoflavones, especially daidzein and genistein are physiologically active substances having an estrogen-like activity, which are notable for an effect of preventing breast cancer or osteoporosis. Moreover, there are also substances which attract attention as powerful antioxidation substances, such as 6,7,4′-trihydroxy isoflavone, 7,8,4′-trihydroxy isoflavone and the like. On the other hand, there was a case that a problem of sterilization of livestock arose when leguminous plants which are rich in isoflavones were used as food for livestock. Therefore, there have been noted the possibility of control of an amount of isoflavones produced in plants for the purpose of improvement in disease resistance of plants and production of isoflavones suitable as feed for livestock or the like. An isoflavone skeleton is biosynthesized via oxidative aryl migration from flavanones to 2-hydroxyisoflavanones, as shown in FIG. 1, and it is known that an enzyme catalyzing the reaction, i.e., 2-hydroxyisoflavanone synthase, exists. Therefore, 2-hydroxyisoflavanone synthase is a very important enzyme in synthesis of isoflavones.
Although isolation and purification of 2-hydroxyisoflavanone synthase, and elucidation of the amino acid sequence of 2-hydroxyisoflavanone synthase and the DNA sequence encoding the amino acid sequence are important for production of isoflavones having the above-mentioned useful activity and control of the amount thereof produced in plants, isolation and purification of 2-hydroxyisoflavanone synthase and elucidation of the cDNA sequence have not been realized.
In order to isolate cDNA of 2-hydroxyisoflavanone synthase and to determine the DNA sequence and the amino acid sequence thereof, the inventors have studied plant materials, culture conditions, mRNA induction, and the like, and as a result, have succeeded in cloning the cDNA encoding 2-hydroxyisoflavanone synthase from the cDNA library produced from the cells obtained at 6 to 12 hours after elicitor treatment of the callus culture of a licorice (Glycyrrhiza echinata) with yeast extracts.